LncRNA LOC102176306 plays important roles in goat testicular development.

Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.

Effect of PPARGC1A on the development and metabolism of early rabbit embryos in vitro.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.

The effects of neuromedin S on the hypothalamic-pituitary-testicular axis in male pigs in vitro.

Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R, LH and FSH mRNAs in anterior pituitary cells, and NMU2R, STAR, P450 and 3ß-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction.

Cloning and mRNA expression of NPB and its effect on hormone secretion of the reproductive cells in the pig.

Neuropeptide B (NPB) is an endogenous ligand for the orphan G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8). Some reports have investigated the role of NPB in the regulation of feeding, energy metabolism and hormone secretion in many species. However, few papers reported the physiological function of NPB in the pig. In this study, we cloned and sequenced the NPB mRNA from a pig, which was found to consist of 123 bases. NPB mRNA expression was detected in central and peripheral tissues by the quantitative fluorescence method. The results showed that NPB mRNA expression was higher in hippocampus, cerebellum, spinal cord, thymus, tonsil, duodenum, cecum, colon, ovary and testis. The distribution of NPB suggested that it may be involved in the regulation of reproductive functions in the pig. Subsequently, the expression and distribution of NPBWR1 and NPBWR2 were found in Leydig cells and ovarian granular cells. We then investigated the direct effect of NPB on pig reproductive cells in vitro. The results showed that different concentrations of NPB (10-12, 10-10, 10-8 and 10-6 M) promoted the secretion of testosterone in Leydig cells in concentration-dependent manner. Different doses of NPB could promote the secretion of progesterone in ovarian granulosa cells in dose-dependent manner. Low concentrations of NPB (10-8 and 10-10 M) promoted estradiol secretion, but high concentrations of NPB (10-6 M) inhibited its secretion. All the results suggested that the NPB/NPBWR1 or NPBWR2 system may play a role in modulating the reproductive activity in the pig.

Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells.

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells.

During goat follicular development, abnormal expression of peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1α) in granulosa cells (GCs) may contribute to follicular atresia with unknown regulatory mechanisms. In this study, we investigate the effect of ectopic expression or interference of PGC-1α on cell apoptosis of goat first passage granulosa cells (FGCs) in vitro. The results indicate that PGC-1α silencing by short hairpin RNA (shRNA) in goat FGCs significantly reduced mitochondrial DNA (mtDNA) copy number (P < 0.05), changed mitochondria ultrastructure, and induced cell apoptosis (P < 0.05). The transcription and translation levels of the apoptosis-related genes BCL-2-associated X protein (BAX), caspase 3, and caspase 9 were significantly up-regulated (P < 0.05, respectively). Moreover, the ratio of BAX/B-cell lymphoma 2 (BCL-2) was reduced (P < 0.05), and the release of cytochrome c (cyt c) and lactate dehydrogenase (LDH) was significantly enhanced (P < 0.05, respectively) in PGC-1α interference goat FGCs. Furthermore, the expression of anti-oxidative related genes superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase (CAT) was down-regulated (P < 0.05, respectively) and the activity of glutathione/glutathione disulfide (GSH/GSSG) was inhibited (P < 0.05). While enforced expression of PGC-1α increased the levels of genes involved in the regulation of mitochondrial function and biogenesis, and enhanced the anti-oxidative and anti-apoptosis capacity. Taken together, our results reveal that lack of PGC-1α may lead to mitochondrial dysfunction and disrupt the cellular redox balance, thus resulting in goat GCs apoptosis through the mitochondria-dependent apoptotic pathway.

Heat-stress-induced metabolic changes and altered male reproductive function.

Heat stress can cause systemic physiological and biochemical alterations in living organisms. In reproductive systems, heat stress induces germ cell loss and poor quality semen. However, until now, little has been known about such a complex regulation process, particularly in the perspective of metabolism. In this study, serum, hypothalamus, and epididymis samples derived from male SD (Sprague-Dawley) rats being exposed to high environmental temperature (40 °C) 2 h per day for 7 consecutive days were analyzed using metabonomics strategies based on GC/TOFMS. Differentially expressed metabolites reveal that the energy metabolism, amino acid neurotransmitters, and monoamine neurotransmitters pathways are associated with heat stress, in accordance with changes of the three upstream neuroendocrine system pathways in the SNS (sympathetic adrenergic system), hypothalamic pituitary adrenal axis (HPA), and hypothalamic pituitary testis axis (HPT) axis. Many of these metabolites, especially in the epididymis, were found to be up-regulated, presumably due to a self-preserving action to resist the environmental hot irritation to maintain normal functioning of the male reproductive system.

Effects of central and peripheral administration of neuropeptide s on the level of serum proinflammatory cytokines in pigs.

OBJECTIVES: The recently discovered neuropeptide S (NPS) and its cognate receptor represent a novel system of neuromodulation and are involved in many physiological and pathological processes. NPS has been implicated in the regulation of proinflammatory cytokine secretion in the pulmonary alveolar macrophages (PAMs) of pigs in vitro. In this study, we tested the hypothesis whether either central or peripheral injection of NPS would stimulate the secretion of the proinflammatory cytokines in pigs. METHODS: In experiment 1, pigs were fitted with an intracerebroventricular cannula and indwelling jugular catheters, and were then randomly assigned to receive 10 or 30 nmol NPS in artificial cerebrospinal fluid. In experiment 2, pigs were fitted with indwelling jugular catheters, and randomly received 15 or 30 nmol NPS in saline. Serial blood samples were collected every 10 min for 1 h before and for 2 h after injections, and serum concentrations of IL-1ß, IL-6 and TNF-α were determined. RESULTS: Serum concentrations of these cytokines were increased in pigs that received central and peripheral injection of NPS, and the elevated secretion of these cytokines was in a time- and concentration-dependent manner. CONCLUSION: The level of serum proinflammatory cytokines could be activated by both central and peripheral administration of NPS in a dose- and time-dependent manner in the pig. The present data support the concept that NPS may be considered as a potent modulator for the immune system and may play an important role in the inflammation and immune system of pigs.

Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

AIMS: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). METHODS: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. RESULTS: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. CONCLUSION: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Development of an indirect ELISA with artificially synthesized N protein of PPR virus.

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 µg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.

Effect of rifaximin on gut-lung axis in mice infected with influenza A virus.

Gut-lung axis injury is a common finding in patients with respiratory diseases as well as in animal model of influenza virus infection. Influenza virus damages the intestinal microecology while affecting the lungs. Rifaximin, a non-absorbable derivative of rifamycin, is an effective antibiotic that acts by inhibiting bacterial RNA synthesis. This study aimed to determine whether rifaximin-perturbation of the intestinal microbiome leads to protective effects against influenza infection, via the gut-lung axis. Our results showed that influenza virus infection caused inflammation of and damage to the lungs. The expression of tight junction proteins in the lung and colon of H1N1 infected mice decreased significantly, attesting that the barrier structure of the lung and colon was damaged. Due to this perturbation in the gut-lung axis, the intestinal microbiota became imbalanced as Escherichia coli bacteria replicated opportunistically, causing intestinal injury. When influenza infection was treated with rifamixin, qPCR results from the gut showed significant increases in Lactobacillus and Bifidobacterium populations, while Escherichia coli populations markedly decreased. Furthermore, pathology sections and western blotting results illustrated that rifaximin treatment strengthened the physical barriers of the lung-gut axis through increased expression of tight junction protein in the colon and lungs. These results indicated that rifaximin ameliorated lung and intestine injury induced by influenza virus infection. The mechanisms identified were the regulation of gut flora balance and intestinal and lung permeability, which might be related to the regulation of the gut-lung axis. Rifaximin might be useful as a co-treatment drug for the prevention of influenza virus infection.

Arginine infusion rescues ovarian follicular development in feed-restricted Hu sheep during the luteal phase.

The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 µmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.

Suppression of miR-1197-3p attenuates H2O2-induced apoptosis of goat luteinized granulosa cells via targeting PPARGC1A.

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) acts as a powerful coactivator of many transcriptional factors that relate to granulosa cell (GC) apoptosis. In this study, the miRNAs mediating goat follicular atresia and luteinized granulosa cell (LGC) apoptosis induced by hydrogen peroxide (H2O2) via PPARGC1A were investigated. Our results showed that miR-1197-3p targeted PPARGC1A was predicted by bioinformatics algorithm and verified by luciferase reporter assay. In addition, miR-1197-3p promoted goat LGC apoptosis via PPARGC1A through mitochondrial-dependent apoptosis pathway, and these effects could be restored by PPARGC1A overexpression. Moreover, H2O2-induced LGC apoptosis significantly upregulated miR-1197-3p expression and downregulated PPARGC1A level. Pretreatment of miR-1197-3p inhibitor alleviated LGC apoptosis induced by 400 µM H2O2 for 12 h, and preserved the mitochondrial membrane potential by increasing PPARGC1A expression. In conclusion, miR-1197-3p might act as an essential regulator of goat LGC apoptosis potentially via the mitochondrial-dependent apoptosis pathway by targeting PPARGC1A.

Expression of Hippo signaling pathway components in Hu sheep male reproductive tract and spermatozoa.

Hippo signaling pathway is essential for tissue development and homeostasis, while its specific role in male reproductive tract development is still unclear. The objective of this study is to elucidate the localization and expressions of key Hippo pathway components (MST1/2, LATS1/2 and YAP1) in male reproductive tract (testis, epididymis, and ductus deferens) of prepubertal (3-month-old) and postpubertal (9-month-old) Hu sheep, as well as in the cauda epididymal and ejaculated spermatozoa. Results showed that the Hippo pathway proteins were diversely localized in male reproductive tract portions, and most of their expression levels increased during sheep testicular maturation. In addition, these Hippo components were mainly localized and highly expressed in ejaculated spermatozoa compared with cauda epididymal spermatozoa. In ejaculated spermatozoa, LATS1 was localized in the acrosomal head region, and LATS2 and YAP1 were expressed in the midpiece part. Taken together, the presence of Hippo signaling cascade in the pubertal development of male reproductive tract and spermatogenesis of Hu sheep, provides new insights on the function of these components in the process of male sexual maturation, capacitation and fertilization.

Distribution of orexin B and its relationship with GnRH in the pig hypothalamus.

Increasing evidence suggests that orexins--hypothalamic neuropeptides--act as neurotransmitters or neuromediators in the brain, regulating autonomic and neuroendocrine functions. Orexins are closely associated with gonadotropin-releasing hormone (GnRH) neurons in the preoptic area and alter luteinizing hormone (LH) release, suggesting that they regulate reproduction. Here, we investigated the distribution of orexin B (immunohistochemical technique) and the relationship between orexin B and GnRH containing fibres and neurons in the pig hypothalamus using double immunofluorescence and laser-scanning confocal microscopy. Orexin B immunoreactive neurons were mainly localized in the perifornical area (PeF), dorsomedial hypothalamic nucleus (DMH), zona incerta (ZI) and the posterior hypothalamic area (PH), with a sparser distribution in the preoptic and anterior hypothalamic area. Immunoreactive fibres were distributed throughout the central nervous system. Approximately 30% GnRH neurons were in close contact with orexin B immunoreactive fibres, among these approximately 6% of GnRH neurons co-localized with orexin B perikarya in the region between the caudal preoptic area and the anterior hypothalamic area. Orexin B may regulate reproduction by altering LH release in the hypothalamus.

Distribution of the pig gastrin-releasing peptide receptor and the effect of GRP on porcine Leydig cells.

Gastrin-releasing peptide (GRP) is a mammalian bombesin (BN)-like peptide which plays a role in a number of important physiological functions via its receptor (gastrin-releasing peptide receptor, GRPR) in most animals. However, little is known about the gene encoding GRPR and its functions (especially reproduction) in pigs. In this study, we first cloned and analyzed the pig GRPR cDNA. Then we systematically investigated the expression levels of GRPR mRNA by relative real-time PCR (RT-PCR), and analyzed the distribution of the GRPR protein in pig tissues via immunohistochemistry (IHC). Finally, we studied the effect of GRP on testosterone secretion and GRPR (mRNA and protein) expression in Leydig cells. Results showed that the pig GRPR cDNA cloned at 1487bp, including one open reading frame (ORF) of 1155bp and encodes 384 amino acids. Significantly, compared with other species, the cDNA sequence and amino acid sequence of the pig GRPR were highly homologous and conservative. The RT-PCR results showed that: in the central nervous system (CNS) and the pituitary, GRPR mRNA was found in the cerebellum, hypophysis, spinal cord and hypothalamus; in the peripheral tissues, GRPR mRNA was mainly expressed in the pancreas, esophagus, ovary, testis, spleen, thymus, jejunum lymph node, muscle and fat. Moreover, the IHC results showed that GRPR immunoreactivity was widely distributed in the pig tissues and organs, such as the pancreas, esophagus, testis, ovary, spleen, pituitary gland and adrenal gland. In addition, we found that GRP promotes testosterone secretion, and increases GRPR mRNA and protein expression in cultured Leydig cells in vitro. These molecular and morphological data not only describe the anatomical locations of GRPR in pigs, but also provide the theoretical foundation for further research into its possible physiological functions in pigs. These results suggest that the GRP/GRPR system may play an important role in regulating the reproductive system of the boar.

Effects of neuromedin B on steroidogenesis, cell proliferation and apoptosis in porcine Leydig cells.

Neuromedin B (NMB), a mammalian bombesin-related peptide, has numerous physiological functions, including regulating hormone secretions, cell growth, and reproduction, by binding to its receptor (NMBR). In this study, we investigated the effects of NMB on testosterone secretion, steroidogenesis, cell proliferation, and apoptosis in cultured primary porcine Leydig cells. NMBR was mainly expressed in the Leydig cells of porcine testes, and a specific dose of NMB significantly promoted the secretion of testosterone in the primary Leydig cells; moreover, NMB increased the expression of mRNA and/or proteins of NMBR and steroidogenic mediators (steroidogenic acute regulatory (STAR), CYP11A1, and HSD3B1) in the Leydig cells. In addition, specific doses of NMB promoted the proliferation of Leydig cells and increased the expression of proliferating cell nuclear antigen and Cyclin B1 proteins, while suppressing Leydig cell apoptosis and decreasing BAX and Caspase-3 protein expression. These results suggest that the NMB/NMBR system might play an important role in regulating boar reproductive function by modulating steroidogenesis and/or cell growth in porcine Leydig cells.

Metabonomics Approach to Assessing the Modulatory Effects of Kisspeptin-10 on Liver Injury Induced by Heat Stress in Rats.

The protective effects of Kisspeptin on heat-induced oxidative stress in rats were investigated by using a combination of biochemical parameters and metabonomics. Metabonomic analyses were performed using gas chromatography/mass spectrometry in conjunction with multivariate and univariate statistical analyses. At the end point of the heat stress experiment, histological observation, ultrastructural analysis and biochemical parameters were measured. Metabonomic analysis of liver tissue revealed that Kisspeptin mainly attenuated the alteration of purine metabolism and fatty acid metabolism pathways. Futhermore, Kisspeptin also increased the levels of GSH, T-AOC as well as SOD activities, and upregulated MDA levels. These results provide important mechanistic insights into the protective effects of Kisspeptin against heat-induced oxidative stress.

The effects of kisspeptin-10 on serum metabolism and myocardium in rats.

Kisspeptin is a peptide encoded by the Kiss 1 gene and is also called metastin. Previous studies have generally focused on several functions of this peptide, including metastasis, puberty, vasoconstriction and reproduction. However, few studies have focused on the cardiac functions of kisspeptin. In the present study, cardiac histomorphology was observed via TEM (transmission electron microscope) and HE and Masson staining to observe instinctive changes. Serum metabolites levels were also measured and analyzed using GC/TOF-MS after injection with kisspeptin-10. A gene chip was employed to screen the potential genes and pathways in the myocardium at the transcriptional leve, while RT-PCR and Western Blot were conducted to verify the relevant mRNA and protein expression, respectively. Histopathological findings demonstrated that there were many irregular wavy contractions through HE staining and increased fibrosis around the heart cells through Masson staining after treatment with kisspeptin-10. Additionally, the main changes in ultrastructure, including changes in mitochondrial and broken mitochondrial cristae, could be observed with TEM after treatment with kisspeptin-10. The PCA scores plot of the serum metabolites was in the apparent partition after injection of kisspeptin-10. Twenty-six obviously changed metabolites were detected and classified as amino acids, carbohydrate metabolites, organic acids and other metabolites. Furthermore, gene chip analysis showed 1112 differentially expressed genes after treatment with kisspeptin-10, including 330 up-regulated genes and 782 down-regulated genes. These genes were enriched in several signaling pathways related to heart diseases. The RT-PCR result for ITGB8, ITGA4, ITGB7, MYL7, HIF1-α and BNP corresponded with the gene chip assay. Moreover, the upregulated genes ITGB8, ITGA4 and BNP also displayed consistent protein levels in Western Blot results. In summary, these findings suggest that kisspeptin-10 could alter the morphology and structure of myocardial cells, serum metabolite levels, and expression of genes and proteins in heart tissues. Our work determined the profound effects of kisspeptin-10 on the heart, which could further lead to the development of therapeutics related to kisspeptin-10, including antagonists and analogs.

Cloning and expression patterns of neuromedin U and its receptors in pigs.

Neuromedin U (NMU) is a highly conserved neuropeptide that performs a variety of physiological functions in animals via neuromedin U receptor-1 (NMUR1) and neuromedin U receptor-2 (NMUR2). In this study, we cloned the pig NMU, NMUR1 and NMUR2 genes. Bioinformatics analysis demonstrated that the pig NMU cDNA encoded the amino acids Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2 at the C-terminus and that the NMU receptors, which are G-protein-coupled receptors (GPCRs), contained the seven transmembrane domains typical of GPCRs. Systemic NMU, NMUR1 and NMUR2 mRNA expression was investigated in various pig tissues using real-time RT-PCR. NMU mRNA was expressed both in the central nervous system (CNS) and in peripheral tissues. NMUR1 mRNA was widely expressed in peripheral tissues, whereas NMUR2 mRNA was mainly expressed in the CNS. Immunohistochemistry (IHC) was used to determine the expression patterns of NMU and NMUR1, which were predominantly located in the gastrointestinal tract, genitourinary organs, and immune organs. This study presents molecular and morphological data to aid in additional NMU research in pigs.